애기장대 배 발달과정 동안 DNA 메틸레이션 역동성과 그 조절의 중요성

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 생명과학부, 2023. 8. 최연희.DNA methylation refers to a modification at the C-5 of a cytosine base. DNA methylation is one of the epigenetic factors which can alter gene and transposon activity without changing DNA sequences. It also helps organisms for adapting to their environments. A double fertilization takes place in flowering plants; a sperm cell fused with an egg cell produces diploid embryo and the other sperm cell fused with diploid central cell produces triploid endosperm. Endosperm nourishes developing embryo, thus it has a similar function to a placenta in mammals. Different from embryo, endosperm is a terminally differentiated companion tissue in that its genome is not transmitted to the next generation. Embryo and endosperm develop inside the seed coat. As seed matures, growing embryo takes the most of the seed volume at the later stage of embryogenesis. A lot of studies reported the defects in DNA methylation mutants embryogenesis, thereby it has been well known that the proper DNA methylation is critical for normal embryo development. However, the causality of methylation difference between different Arabidopsis ecotypes during embryo development has not been fully understood. Here, I present comprehensive pairwise comparisons for the methylation differences during seed ripening and germination between two different ecotypes. Using Columbia-0 (Col-0) and Cape Verde Island (Cvi) ecotypes, I chose three developmental seed stages; 1) freshly harvested (FH), 2) after ripening (AR), 3) germination stimulated (GS). I discovered overall dynamics of DNA methylation during seed ripening and germination. Among three cytosine contexts, CG context showed the most differently methylated as Col displayed always higher mCG levels than Cvi for three stages in both genic regions and transposable elements (TEs). In contrast, non-CG methylation levels were very similar between both ecotypes. Interestingly, I found Col-specific DNA sequences (Col-own region, COR), where were considered as diverged regions, were more hypermethylated than conserved regions (CR) in both ecotypes. CORs were enriched with TEs and structural variations (SVs) including insertions, deletions. TEs in COR were longer in size, more pericentromeric, and more hypermethylated than those in the CRs, suggesting that the COR TEs are younger than TEs in CR. By using previously reported ago4/6/9 and drm1/2 methylome datasets, I demonstrated that RNA-directed DNA methylation (RdDM) pathway contributes to the hypermethylation in younger CORs. Although the representative patterns of DNA methylation, specifically for a gradual increase in CHH methylation during embryo development have been already reported by other groups, by using different five embryo developmental stages, I found nine different clustered regions based on the similar methylation change during embryonic development. Those clusters showed different positional and chromatic characters. Taken all together, my results demonstrate the possibility of regulation on newly acquired regions by RdDM pathway. And I suggest a new analysis to reveal the biological meanings of CHH methylation changes during embryo development.DNA 메틸화는 사이토신 염기의 5번 탄소에 생긴 변형을 의미한다. DNA 메틸화는 DNA 서열을 바꾸지 않고 유전자와 전위인자 활성을 변화시킬 수 있는 에피유전학적 요인 중 하나이다. 또한 유기체가 그들의 환경에 적응하는 것을 돕는다. 난세포와 융합된 수배우자는 이배체 배를 형성하고, 다른 수배우자는 이배체 중심세포와 융합되어 삼배체 배유를 형성한다. 배유는 발달 중인 배에 영양을 공급하기 때문에 포유류의 태반과 비슷한 기능을 한다. 배와 달리 배유는 유전체가 다음 세대로 전달되지 않는다는 점에서 분화된 동반 조직이다. 배와 배유는 종자껍질 안에서 발달한다. 종자가 성죽함에 따라, 성장하는 배는 발생 후기 단계에서 종자 부피의 대부분을 차지한다. 많은 연구들이 DNA 메틸화 돌연변이 발생시 결함을 보고했고, 따라서 적절한 DNA 메틸화가 정상적인 배 발달에 중요하다는 것은 잘 알려져 있다. 그러나 배 발달 중 다른 애기장대 생태형 간 메틸화 차이의 인과관계는 완전히 이해가 되지는 않았다. 이 논문에서 종자가 성숙하고 발아하는 동안 두 생태형 사이의메틸화 차이를 포괄적인 상호비교를 제시한다. Columbia-0(Col-0)와 Cape Verde Island(Cvi) 생태형을 사용하여 3 단계의 종자 발달 단계를 정했다; 1) 숙성 전(FH), 2) 숙성 후(AR), 3) 발아 자극처리(GS). 종자가 성숙하고 발아하는 동안 DNA 메틸화의 전반적인 변화를 발견했다. 3개의 사이토신 종류 중 가장 다르게 메틸화된 점은 유전자 영역과 전위인자에서 3단계 모두 Col-0가 항상 Cvi보다 높은 mCG 수준을 보인다는 것이다. 대조적으로, CG이외의 메틸화 수준은 두 생태형이 매우 유사했다. 흥미롭게도, 분화된 영역으로 간주되는 Col-0 특이적 DNA 서열(COR)이 두 생태형 모두에서 보존 서열(CR) 보다 더 메틸화가 되었다는 것을 발견됐다. COR은 전위인자를 다수 포함하며 삽입, 삭제를 포함한 구조 변이(SV)가 많았다. COR의 전위인자가 CR의 전위인자보다 애기장대 유전체에 늦게 도입됐다는 점을 시사한다. 이전에 보고된 ago4/6/9 및 drm1/2 메틸롬 데이터를 사용하여 RdDM pathway가 COR의 높은 메틸화 수준에 기여한다는 것을 입증했다. 비록 DNA 메틸화의 대표적인 패턴, 특히 배 발달 동안 CHH 메틸화의 점진적인 증가가 다른 그룹을 통해 이미 보고 되었지만, 5 단계의 배 발달 과정단계의 배를 사용하여 메틸화 변화를 기준으로 9개의 서로 다른 클러스터 지역을 발견했다. 그 클러스터들은 유전체상 위치와 염색질의 특성이 다르게 나타났다. 요약하면, 본 연구는 RdDM pathway에 의해 새로 획득된 DNA서열에 대한 조절의 가능성을 시사한다. 그리고 배 발달 중 CHH 메틸화 변화의 생물학적 의미를 밝히기 위한 새로운 분석을 제안한다.Abstract 1 Table of Contents 5 List of Figures 9 List of Tables 12 Abbreviations 13 I. Background 14 1.1 Epigenetics 14 1.2 DNA methylation in Arabidopsis 15 II. Ecotype specific DNA methylation 18 2.1 Introduction 18 2.2 Materials and methods 21 2.2.1 Plant growth condition 21 2.2.2 DNA library construction 21 2.2.3 Sequencing data processing 22 2.2.4 Measurement of DNA methylation levels 22 2.2.5 Identification of differentially methylated region 23 2.2.6 Identification of Conserved and Col-0 own region 23 2.2.7 Methylation of genebody and transposable element 23 2.2.8 Identification of variable regions between ecotypes 24 2.3 Results 25 2.3.1 CG eDMRs are strongly maintained in the genebody throughout development 25 2.3.2 Methylation levels on eDMRs are positively correlated with differential expression of 24-nt small RNAs 34 2.3.3 A large composition of TE and intrinsic features in COR contribute to hypermethylation 40 2.3.4 The correlation between mC levels and SNPs in COR 44 2.3.5 Ecotype-specific structural variations in COR are hypermethylated 50 2.4 Discussion 57 2.4.1 DNA methylation during seed ripening and germination 57 2.4.2 Ambiguous cause and function of CG methylation levels 57 2.4.3 COR was not lost in Cvi, but acquired in Col-0 58 2.4.4 DNA methylation of newly acquired gene 62 III. DNA methylation during embryo development 63 3.1 Introduction 63 3.2 Materials and methods 65 3.2.1 Plant growth condition 65 3.2.2 Construction of bisulfite sequencing libraries 65 3.2.3 WGBS data processing and DNA methylation level 66 3.2.4 Clustering 66 3.2.5 Genomic features of clusters 67 3.2.6 Distributions of each cluster on chromosome 67 3.2.7 Cytosine frequency of predicted de novo LTR 67 3.3 Results 69 3.3.1 Dynamic change of CHH methylation during embryo development 69 3.3.2 The regions with different patterns of change during embryo development 74 3.3.3 The composition of genomic features and distribution on chromosome by a class 79 3.4 Discussion 83 IV. References 87 V. Abstract in Korean 국문초록 94박

Analyzing Improvements under the Construction Technology Promotion Act

학위논문(석사) -- 서울대학교대학원 : 공과대학 건축학과, 2024. 2. Changbum Ryan Ahn.An Examination of Reporting and Recording Practices in Construction Safety Management: Analyzing Improvements under the Construction Technology Promotion Act Jaehoon Lee Department of Architecture & Architectural Engineering The Graduate School Seoul National University With the increasing frequency and severity of accidents in the construction industry, the importance of effective safety management is being emphasized more than ever. Despite the government's efforts to mitigate industrial accidents through substantial budget allocations and legislative enactments, the construction industry continues to face a significant number of fatalities each year. A major issue in the current safety management practice of construction industry is the excessive documentation required at construction sites. This excessive documentation leads safety managers to neglect their primary duties of overseeing on-site safety. Although studies have been conducted to improve the construction industry safety management practice, most studies have focused on institutional improvements. These studies have not adequately addressed the real reasons behind safety managers' neglect of their duties, nor have they provided tangible solutions. Additionally, the issuance of documentation manuals that do not reflect the experiences and challenges of on-site personnel has led to a procedural approach to documentation. Furthermore, these efforts have primarily focused on the Occupational Safety and Health Act, resulting in a relatively insufficient understanding of the documentation required by the Construction Technology Promotion To address these gaps, this study focuses on the Construction Technology Promotion Act to diagnose the problems of safety management documentation practices at construction sites, identify the main causes of inefficiency, and propose strategies for improving current documentation practices. Field surveys targeting safety management personnel were conducted to gather opinions on the redundancy, necessity, and costs associated with the current documentation practice. Based on these insights, the study diagnosed the problems of the existing practice and identified documents in need of improvement. Additionally, a verification based on actual documents written at construction sites was conducted to propose strategies for addressing these issues. This study provides a detailed diagnosis of the current safety management documentation practices by reflecting the opinions of construction site documentation personnel. It also expands the understanding of the entire practice by focusing on the Construction Technology Promotion Act. The findings of this study offer crucial information for setting a clear direction for the simplification and improvement of current documentation practices, ultimately contributing to the reduction of construction site accidents and the enhancement of safety management efficiency.건설산업의 사고발생 빈도와 심각성이 증가함에 따라, 건설 안전관리의 중요성이 강조되고 있다. 정부는 산업재해를 경감하기 위해 다량의 예산 편성 및 법령 제정 등 정책적 노력을 하고 있음에도 건설산업은 여전히 매년 상당한 사망자가 발생하고 있는 실정이다. 이와 같은 현행 건설산업 안전관리 관행의 문제점으로 건설현장의 과도한 서류작업이 지적되고 있다. 현장의 과도한 서류작업으로 인해, 안전관리 업무를 수행해야 할 안전관리자들이 본연의 업무를 등한시한 채 서류작업에 전념하고 있는 실정이다. 건설산업 안전관리 체계를 개선하기 위한 연구들이 수행되어왔으나, 대부분의 연구들은 제도 자체의 개선에 중점을 두고 있어 안전관리자들이 현장관리 업무에 소홀하게 되는 실질적 원인에 관한 고찰과 해결방안 제시가 제한된다. 또한, 현장 담당자들의 경험과 고충을 반영하지 못한 서류작업 매뉴얼 발간 등으로 인해 형식적 서류작업 문화를 형성하고 있는 실정이다. 더불어, 이 마저도 산업안전보건법을 위주로 수행되어, 건설기술진흥법에 의한 서류작업에 관한 이해는 상대적으로 미흡한 실정이다. 이에 본 연구는 이러한 격차를 해소하기 위해 건설기술진흥법에 초점을 맞추어 건설현장의 안전 관리 서류 작업 관행의 문제점을 진단하고, 비효율을 초래하고 있는 주요 원인을 도출하였으며, 현행 서류작업 관행의 효율화를 위한 개선전략을 제안하였다. 이를 위해, 안전관리자 및 안전관리담당자 등 건설현장 안전관리 서류작업 담당자 대상 현장조사를 수행하였으며, 현행 서류작업 관행에 관한 중복성 및 필요성, 소요비용 등 현장의 의견을 수렴하였다. 수렴한 현장의 의견을 토대로, 현행 서류작업 관행의 문제점을 진단하였으며, 현행 안전관리 서류작업 관행 상의 한계점과 개선이 필요한 서류를 식별하였다. 더불어, 건설현장에서 작성된 실물 서류기반 검증을 통해 해당 문제점을 개선하기 위한 전략을 제안하였다. 본 연구는 건설현장 서류작업 담당자들의 의견을 반영하여 현행 안전관리 서류작업 관행의 문제점을 심도 있게 분석함으로써, 서류작업 관행의 효율화를 위한 정밀 진단을 제공하였다. 또한, 건설기술진흥법에 초점을 맞춘 분석을 통해 건설현장에서 수행되는 안전관리 서류작업의 관행 전반에 대한 이해를 확장시켰다. 연구의 결과는 현행 안전관리 서류작업 관행의 효율화를 위한 명확한 방향성 설정에 필요한 중요한 정보를 제공하며, 궁극적으로 건설현장 안전사고 저감 및 안전관리 효율 증진에 기여할 수 있을 것으로 기대된다.Chapter 1. Introduction . 1 1. 1 Research Background 1 1. 2 Problem Description 4 1. 3 Research Objectives and Scope 7 Chapter 2. Practical Limitations . 8 2. 1 Preliminary Study on Safety Management Improvements 8 2. 2 Limitations of Current Safety Management Documentation Practice in Korea 10 Chapter 3. Empirical Analysis of Safety Management Documentation in Korean Construction Sites . 13 3. 1 Inventory of Safety Management Documentation 16 3. 2 Redundancy of Safety Management Documentation 19 3. 3 Necessity of Safety Management Documentation 23 3. 4 Cost of Safety Management Documentation 27 3. 5 Evaluation of the Existing Safety Management Documentation Practice 31 3. 6 Summary 36 Chapter 4. Discussions . 38 4. 1 Developing Standardized Official Documentation Forms 38 4. 2 Simplifying Safety Management Documentation Practice 41 Chapter 5. Conclusions . 53 5. 1 Research Results 53 5. 2 Research Contributions 56 Bibliography 57석

산업공정 리그닌을 이용한 약물 방출 조절형 나노입자 합성

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 산림과학부(환경재료과학전공), 2021. 2. 최인규.There has been growing interest in lignin as a promising source for drug delivery system preparation in recent years. The lignin-based nanoparticle has the potential to be preferably used because of its controllable intracellular uptake and stimuli-controlled drug-releasing ability. However, few research investigated the effect of structural characteristics of lignin on the synthesis of nanoparticles and its essential features such as size, colloidal stability, and biocompatibility. This research focuses on how the molecular structures of technical lignin (kraft and alkaline/soda lignin) affect the lignin nanoparticle properties. Based on these results, drug-encapsulated lignin nanoparticles were synthesized, and those drug-releasing efficiencies were investigated through evaluation of in vitro and in vivo biocompatibility. First, lignin fractions with different molecular weights (Mw 1,460 ~ 12,900), phenolic hydroxyl group (total 3.7 ~ 6.2 mmol g-1), and main internal linkages (β-O-4, β-β, and β-5) were obtained by performing a sequential solvent extraction of kraft lignin (KL). Subsequently, spherical lignin nanoparticles (KLNPs, i.d. 193 ~ 1,039 nm) were synthesized by the nanoprecipitation process. The low-molecular-weight KL fractions generated KLNPs with a relatively enlarged diameter but less size-distributed. Further analysis showed that molecular weight, total, and phenolic hydroxyl group content in the lignin highly correlated with nanoparticle size. Every KLNPs exhibited comparable and good colloidal stability (-44.5 ~ -29.1 mV). Cell viability and hemotoxicity assessments revealed higher and sustained biocompatibility of KLNPs even at a high concentration, at least for three days. Second, KL was utilized as a polymer for the drug-encapsulated nanoparticle (ELNP) synthesis. Successful drug encapsulation of ELNPs by nanoprecipitation was determined (max. 59% for coumarin 6 and max. 73% for doxorubicin (DOX)). Compared to KLNP, the size (i.d. 176 ~ 469 nm) and surface charge (-40.5 ~ -32.7 mV) of ELNP showed no remarkable change regardless of introducing the drug in the particle. The drug release profile of ELNPs was faster in the relatively acidic condition (over 70% within 0 ~ 4 h) than the neutral (less than 50% within 0 ~ 4 h). In vitro cytotoxicity assay revealed the drug release effect of ELNPs, delaying but not decreasing the anticancer activity. In addition, the hemocompatibility test, in vivo allergic reaction test, and tumor inhibition assays using mice tumors showed high ELNP biocompatibility and side effect reduction during the chemotherapy. Lastly, alkaline/soda lignin (AL) was sequentially solvent-extracted, or its phenolic hydroxyl groups were chemoselectively methylated. These lignins with the modified structure were handled to form nanoparticles (ALNP) to clarify the effect of the lignin structure on the physical properties of nanoparticles. Pure ALNPs were obtained from neat lignin, solvent-extracted fractions (ALNP-Fs, i.d. 414 ~ 1,214 nm), and methylated lignins (ALNP-Ms, i.d. 516 ~ 721 nm). Specifically, the size properties of ALNP-Ms showed a very high negative correlation (R2 = 0.95) with the phenolic hydroxyl group amount. It indicates that the phenolic hydroxyl groups in lignin are the main structures that affect the nucleation and/or condensation during the nanoprecipitation process. ALNPs exhibited high colloidal stability (-42.3 ~ -32.7 mV), and most of them also showed good in vitro biocompatibility.최근 리그닌을 약물 전달 시스템의 재료로 이용하는 연구가 주목받고 있다. 특히 리그닌으로 나노입자를 제조하는 연구는 약물 수송 및 방출 조절에 관한 장점을 부여할 수 있어 선호되는 추세이다. 하지만 그중 리그닌의 구조적 특성이 나노입자의 형성 과정이나 물성(크기, 콜로이드 안정성, 생체안전성)에 미치는 영향에 주목한 연구는 거의 없는 실정이다. 본 연구에서는 산업공정 부산물로 발생하는 리그닌 부산물의 구조적 특성이 리그닌 나노입자의 물성에 미치는 영향을 이해하고자 하였다. 해당 결과를 바탕으로 약물 저장 리그닌 나노입자를 합성하여 약물 방출 특성과 기대 효과를 생체외 및 생체내 조건에서의 생체안전성 실험을 통하여 구명하였다. 우선 산업공정 부산물인 kraft 리그닌을 순차적 용매 분획화하여 분자량, 수산기 함량, 분자간 주요 결합 함량이 각기 다른 5개 분획으로 분리하였다. 각 리그닌 분획으로부터 THF-리그닌 용액과 증류수의 치환에 의한 나노침전법을 통하여 나노입자를 획득하였으며 입자 크기는 최소 193 nm에서 최대 1,039 nm로 다양하게 나타났다. 리그닌 분자량이 작을수록, 페놀성 수산기 함량이 높을수록 크면서 균질한 분포를 보이는 입자가 형성되었으며 kraft 리그닌의 구조적 특성과 형성된 나노입자의 입자 크기와 분포는 리그닌 분자량과 수산기 함량, 특히 페놀성 수산기 함량에 영향을 받는 것으로 판단되었다. 나노입자는 대체로 깔끔한 구형으로 형성되었으며 증류수에 안정적인 콜로이드 상태로 분포하였다(-44.5 ~ -29.1 mV). 세포 생존율 및 혈구독성 평가 결과 리그닌 나노입자는 높은 농도 조건에서도 최소 3일간 높은 수준의 지속적인 세포안전성을 보이는 것을 확인하였다. 위 결과를 바탕으로 선택한 특정 리그닌 조건으로부터 약물 저장 나노입자를 합성하였으며 약물 방출 조절 효과를 입증하기 위한 실험을 수행하였다. 약물 저장 리그닌 나노입자는 coumarin 6에 대하여 최대 59%, 독소루비신에 대하여 최대 73%의 약물 저장 효율을 나타내었다. 약물 저장 리그닌 나노입자의 크기(176 ~ 469 nm)는 대조군과 비교하였을 때 약물 첨가에 큰 영향을 받지 않는 것으로 나타났다. 또한, 약물 저장 여부는 나노입자의 콜로이드 안정성에도 영향을 미치지 않았다. 약물 저장 리그닌 나노입자는 대체로 0 ~ 4시간 사이에 빠른 약물 방출 경향을 보였고 혈액과 동일한 pH 조건보다는 산성 조건에서의 약물 방출이 더 빠른 것으로 나타났다. 리그닌 나노입자는 생체외 혈구독성 실험과 생체내 알레르기 반응 실험에서 우수한 생체안전성을 나타냈으며, 생체외 세포독성 실험과 생체내 항암효과 검증 실험에서 약물 방출 조절 및 부작용 경감에 의한 사망률 감소 효과를 나타냈다. Alkaline/soda 리그닌의 경우 순차적 용매 분획화와 별도로 페놀성 수산기의 메틸화 반응을 수행하여 분자량의 변화가 상대적으로 적으면서 페놀성 수산기의 함량이 각기 다른 4개 그룹을 추가로 획득하였다. 구조적 변형을 거친 해당 리그닌 그룹으로부터 나노입자를 획득하여 그 물성을 분석한 결과, 분획화 리그닌 유래 나노입자는 kraft 리그닌에서와 유사한 크기 분포 경향을 보였다. 반면, 메틸화 리그닌 유래 나노입자는 페놀성 수산기가 감소함에 따라 크기가 감소하는(721 ~ 516 nm) 뚜렷한 경향을 보였다. 모든 조건에서 나노입자의 콜로이드 안정성은 높게 유지되었으며 생체안전성 역시 대부분 농도 및 시간 조건에서 안전성을 나타냈다. 본 연구에서는 펄프 및 바이오에탄올 생산공정에서 부산물로 발생하는 리그닌의 고부가가치 활용방안으로 약물 방출 조절 나노입자 합성을 제시하였다. 리그닌의 구조적 특성과 나노입자 물성의 상관성을 구명하여 본 연구에서 다루지 않은 다른 리그닌을 이용한 나노입자 제조 및 물성 제어에 있어서도 단초를 제공하였으며, 생체외 및 동물 실험을 통한 리그닌 나노입자의 약물 방출 조절 및 부작용 경감 효과를 검증하여 약물 전달체 원료로써 실제 적용 가능성을 제시하였다.Contents Chapter 1 Introduction 1 1. Background 2 1.1. The concept of lignocellulosic biorefinery 2 1.2. Lignin production from pulping and sugar-based biorefinery 3 1.3. Understanding the lignin structure for further application 6 1.4. Polymeric nanoparticle for biomedical use 8 2. Objectives 12 3. Literature review 15 3.1. Valorization of lignin macromolecules 15 3.1.1. Isolation of lignin from biomass 15 3.1.2. Determination of lignin structure 19 3.1.3. Lignin fractionation 24 3.1.4. Structural functionalization of lignin 28 3.2. Nano-sized carriers for drug delivery 38 3.2.1. Strategies for drug delivery 38 3.2.2. Nanoparticle synthesis 44 3.2.3. Strategies for controlled drug release 48 Chapter 2 Synthesis of pure nanoparticles with solvent-fractionated kraft lignin and evaluation of their biocompatibility 51 1. Introduction 52 2. Materials and methods 54 2.1. Materials 54 2.2. Lignin fractionation 56 2.3. Characterization of fractionated lignin 58 2.4. Synthesis of lignin nanoparticle 62 2.4.1. Pre-dialysis concentration 62 2.4.2. Lignin fractions 64 2.5. Characterization of lignin nanoparticle 65 2.6. Biocompatibility tests 66 2.6.1. CCK-8 assay 66 2.6.2. Hemolysis assay 68 2.7. Statistical analysis 69 3. Results and discussion 70 3.1. Characteristics of lignin fractions 70 3.2. Particle size and morphology 80 3.3. Particle surface charge 89 3.4. Storage stability of the particle in pure water 90 3.5. In vitro cell viability 93 3.6. Hemocompatibility 98 4. Conclusion 100 Chapter 3 In vivo and in vitro evaluation of drug-encapsulated lignin nanoparticle 101 1. Introduction 102 2. Materials and methods 106 2.1. Materials 106 2.2. Animals and ethics 108 2.3. Synthesis of encapsulated lignin nanoparticle 109 2.3.1. Coumarin 6-encapsulated nanoparticle 109 2.3.2. DOX-encapsulated nanoparticle 110 2.4. Characterization of encapsulated nanoparticle 111 2.4.1. Determination of physical properties of nanoparticle 111 2.4.2. Drug loading 112 2.4.3. In vitro release study 113 2.5. Biocompatibility tests 114 2.5.1. CCK-8 assay 114 2.5.2. Hemolysis assay 115 2.5.3. In vivo detection of an allergic reaction 116 2.6. In vivo anticancer efficacy 117 2.7. Statistical analysis 118 3. Results and discussion 119 3.1. Particle size and encapsulation efficiency 119 3.1.1. Coumarin 6-encapsulated nanoparticle 119 3.1.2. DOX-encapsulated nanoparticle 124 3.2. Particle surface charge 128 3.3. In vitro drug release 130 3.4. In vitro cytotoxicity 135 3.5. Hemocompatibility 138 3.6. Hypersensitivity test 140 3.7. In vivo antitumor efficacy 142 4. Conclusion 145 Chapter 4 Effect of chemoselective methylation of the phenolic hydroxyl group on lignin nanoparticle synthesis 147 1. Introduction 148 2. Materials and methods 150 2.1. Materials 150 2.2. Functionalization of lignin 152 2.3. Characterization of lignin 154 2.4. Synthesis of lignin nanoparticle 156 2.4.1. Pre-dialysis concentration 156 2.4.2. Functionalized lignin 158 2.5. Characterization of lignin nanoparticle 159 2.6. Biocompatibility test 160 2.7. Statistical analysis 161 3. Results and discussion 162 3.1. Characteristics of functionalized lignin 162 3.1.1. Lignin fractions 162 3.1.2. Lignin methylation 174 3.2. Effect of lignin characteristics on nanoparticle size 178 3.2.1. Effect of solvent fractionation 178 3.2.2. Effect of the hydroxyl group 186 3.3. Particle surface charge 190 3.4. Storage stability of nanoparticle 192 3.5. In vitro cell viability 195 4. Conclusion 198 Chapter 5 Conclusion 199 References 203 초록 225Docto

Therapeutic Effects of HIF-1α on Bone Formation around Implants in Diabetic Mice Using Cell-Penetrating DNA-Binding Protein

Patients with uncontrolled diabetes are susceptible to implant failure due to impaired bone metabolism. Hypoxia-inducible factor 1α (HIF-1α), a transcription factor that is up-regulated in response to reduced oxygen during bone repair, is known to mediate angiogenesis and osteogenesis. However, its function is inhibited under hyperglycemic conditions in diabetic patients. This study thus evaluates the effects of exogenous HIF-1α on bone formation around implants by applying HIF-1α to diabetic mice and normal mice via a protein transduction domain (PTD)-mediated DNA delivery system. Implants were placed in the both femurs of diabetic and normal mice. HIF-1α and placebo gels were injected to implant sites of the right and left femurs, respectively. We found that bone-to-implant contact (BIC) and bone volume (BV) were significantly greater in the HIF-1α treated group than placebo in diabetic mice (p < 0.05). Bioinformatic analysis showed that diabetic mice had 216 differentially expressed genes (DEGs) and 21 target genes. Among the target genes, NOS2, GPNMB, CCL2, CCL5, CXCL16, and TRIM63 were found to be associated with bone formation. Based on these results, we conclude that local administration of HIF-1α via PTD may boost bone formation around the implant and induce gene expression more favorable to bone formation in diabetic mice.ope

18F]Mefway 양전자방출단층촬영술로 측정한 갑상선기능저하증이 쥐 뇌의 세로토닌 1A 수용체에 미치는 영향

Dept. of Medicine/박사This study investigated the effects of hypothyroidism on the brain serotonin (5-HT) 1A receptor using animal disease models and 4-(trans-[18F]fluoranylmethyl)-N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexane-1-carboxamide ([18F]Mefway) positron emission tomography (PET) imaging. Five surgically thyroidectomized male Sprague-Dawley rats were assigned to the hypothyroidism group, and five sham-operated male Sprague-Dawley rats were assigned to the control group. Hypothyroid status was confirmed by thyroid function tests. After anesthesia with 2.0% isoflurane in oxygen, fluconazole was infused at a constant rate for one hour to prevent defluorination of the radioligand. Then [18F]Mefway of 8.6–11.1 MBq was administered at a rate of 1 ml/min and dynamic PET scans were performed over the course of 120 min. PET data were reconstructed in user-defined time frames using a two-dimensional ordered-subset expectation maximization algorithm. All PET data were spatially normalized to T2-weighted magnetic resonance imaging templates, and then time-activity curves of the hippocampus, septum, and cerebellum were extracted using predefined volume of interest templates. Non-displaceable binding values in the hippocampus and septum were calculated using a multilinear reference tissue model, with the cerebellum as the reference tissue, and a ligand-specific parametric map was constructed. Time-activity curves revealed that the hippocampal and septal uptakes in the hypothyroidism group were 25–52% higher than those in the control group, and non-displaceable binding potentials in the same regions of thyroidectomized rat brains were about 30% higher than those in controls. In conclusion, hypothyroidism increased the density of postsynaptic 5-HT1A receptors in the hippocampus and septum. Upregulation of postsynaptic 5-HT1A receptors may be responsible for the increased radioligand uptake as an early response to the reduced synaptic concentration of 5-HT caused by hypothyroidism. 이 연구는 동물 질병 모델 및 4-(trans-[18F]fluoranylmethyl)-N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexane-1-carboxamide ([18F]Mefway) 양전자방출단층촬영술(PET)을 사용하여 갑상선기능저하증이 뇌의 세로토닌 1A 수용체에 미치는 영향을 연구하였다. 갑상선절제술을 받은 Sprague-Dawley 수컷 쥐 5마리를 갑상선기능저하증 집단에 배정하였고, 모의 수술(sham operation)을 받은 Sprague-Dawley 수컷 쥐 5마리를 대조군으로 설정하였다. 갑상선기능저하 상태는 갑상선 기능 검사로 확인하였다. 산소에 2%의 아이소플루레인을 혼합하여 마취를 시킨 후, [18F]Mefway의 탈불소화를 방지하기 위하여, 1시간 동안 플루코나졸을 투여하였다. 그 다음 8.6–11.1 MBq의 [18F]Mefway를 1 ml/min 속도로 투여한 후, PET 영상을 120분간 연속적으로 획득하였다. PET 영상은 2차원 정칙화된 기댓값 최대화 알고리즘(ordered subsets expectation maximization algorithm)을 이용하여 재구성하였다. 모든 PET 영상은 T2 강조 자기공명영상 틀(template)에 공간정규화 한 후 사전 정의된 관심영역 틀을 이용하여 해마, 사이막(septum), 소뇌의 시간방사능곡선을 추출하였다. 해마와 사이막의 수용체 결합능(non-displaceable binding potential)은 소뇌를 참조조직으로 삼아 다중선형 참조조직 모형(multilinear reference tissue model)을 통하여 계산하였고 리간드-특정 모수지도(parametric map)를 작성하였다. 시간방사능곡선에서 갑상선기능저하증 집단의 해마와 사이막 섭취는 대조군보다 25-52% 더 많았고 이 부위의 결합능은 갑상선절제술을 받은 쥐에서 대조군에 비하여 30% 가량 더 높았다. 결론적으로, 갑상선기능저하증은 해마와 사이막의 시냅스후 세로토닌 1A 수용체 밀도를 증가시켰다. 이는 갑상선기능저하증으로 인해 시냅스의 세로토닌 농도가 감소하고 이에 따른 초기 반응으로써 시냅스후 세로토닌 1A 수용체가 상향조절 되었기 때문일 것이다.ope

Hormone Therapy in the Era of the COVID-19 Pandemic: A Review

The coronavirus disease 2019 (COVID-19) pandemic has impacted the medical, social, and reproductive health of millions of people since its outbreak. The causative virus transmits, reproduces, and manifests through the respiratory tract. COVID-19 can invade any system of the body, including the cardiovascular and endocrine systems, through a secondary immune response. In particular, because the fatality rate is high in those over the age of 50 years, special attention is required during the medical care of this population. However, considering the benefit of therapy and the risk of COVID-19, high-quality evidence regarding individualized management in relation to hormone therapy is still insufficient in the field of gynecology. Furthermore, this review aims to serve as a reference for clinical application by analyzing and summarizing the results of studies reported to date regarding female hormone therapy in the context of the COVID-19 pandemic.ope

Experimental Investigation of Reverbed Signal Due to Microbubbles within Ship Wake

Microbubbles are created in copious quantities in the wakes of ocean vessels and, because of their large acoustical cross sections, are responsible for the scattering acoustical signature of the wake. The acoustic characteristics of the suspended microbubbles can be used to detect and track the ship wake. In this thesis, we show that the experimental result performed at Korea Maritime University. It was calculated the volume scattering strength via experimental data. We can investigate the motion and distribution of bubble plume within ship wake via volume scattering strength.목차 Ⅰ 그림 목차 Ⅱ 표 목차 Ⅳ Abstract Ⅴ 1. 서 론 1 1.1. 연구의 목적 및 필요성 1 1.2. 연구의 내용 및 방법 3 1.3. 논문의 구성 4 2. 항적에 의한 음향산란특성 5 2.1. 체적산란강도 5 2.2. Wavelet 변환에 의한 난류 유동 특성 10 3. 항적에 의한 음향산란 해상실험 13 3.1. 실험환경 13 3.2. 실험 장비의 구성 16 3.3. 실험조건 및 방법 17 4. 실험결과 분석 19 4.1. 체적산란강도 19 4.1.1. 분석방법 19 4.1.2. 분석결과 23 4.2. Wavelet 변환에 의한 난류 유동 특성 39 4.2.1. 분석방법 39 4.2.2. 분석결과 41 5. 결 론 47 참고 문헌 4

Difficult intubation: lessons learned from the courts of South Korea

ope

Therapeutic use of mesenchymal stem cell-derived extracellular vesicles in acute lung injury

ope

Complications related to cantilever lengths in implant-supported single crowns

Prosthesis using implant has become a popular modality to treat edentulous patients for the last 35 years. The progress in surface treatment reduces the failure rate and provides better functional and esthetic restorations in dentistry. The earliest form of implant prosthesis was the splinted-fixed type. After full arch and partial edentulous prosthesis began to demonstrate s stable clinical successes rate, single crowns started to be restored with implants.When the implant supported single crown is located most distally in the arch, special biomechanical factors should be considered. Especially when the implant is placed in a more distal position, there will be exposure to tremendous lateral forces resulting in possible complications. The purpose of this study is to analyze the relationship between complications and distance from adjacent teeth to the implant, and to identify the types and frequency of complications. A total of 115 patients charts were investigated randomly from the implant clinic at Ewha WomansUniversity Hospital and Yonsei University Dental Hospital. The patients received an implant supported single crown from 1996 to 2007 and annual afterwards. Patients were grouped according to the presence or lack of complications and the relationship of tooth to implant distance and complications was investigated. Age, sex, and abutment connection type were also analyzed for relationship with complications. The measured data from the two groups were analyzed statistically with SAS version 9.1 (SAS Inc., USA). There were significant statistical differences using logistic analysis (p<0.05) between the tooth to implant distance and complications. The odds ratio was 2.1 and cut off value from maximum value of specificity and sensitivity was 3.39 mm. SPSS 12.0(SPSS Inc. Chicago, Illinois. USA)ope